CRIPTO-1 (CR-1, human; Cr-1, mouse) also known as teratocarcinoma-derived growth factor-1 (U.S. Pat. Nos. 5,792,616; 5,256,643; 5,654,140), and CRIPTO-3 (CR-3, human; Cr-3, mouse) (U.S. Pat. Nos. 5,264,557; 5,620,866; 5,650,285), collectively referred to herein as CRIPTO, are EGF-related proteins. Their genes are expressed in the developing embryo, in normal adult tissues and in tumor cells, including but not limited to, breast cancer cells and colon cancer cells.
It is a discovery of the present invention that CRIPTO expressed in mammalian cells is modified with O-linked fucose, an unusual form of O-linked glycosylation, at amino acid residue threonine-88 of CRIPTO polypeptide as depicted in FIG. 1. Enzymes that add fucose modifications to proteins, recognize a seven amino acid configuration (“fucosylation site”). It is a discovery of the present invention that two residues in particular at positions N−1 (i.e. glycine residue 86 from CRIPTO polypeptides shown in FIG. 1) and N−2 (glycine residue 87 from CRIPTO polypeptides shown in FIG. 1) are required for fucosylation of threonine-88 to occur. Morever, it is a discovery of the present invention that non-fucosylated forms of CRIPTO which act as functional antagonists have anti-tumor activity. Non-fucosylated forms of CRIPTO that can act as functional antagonists include but are not limited to mutant CRIPTO polypeptides which alter the binding of CRIPTO to a CRIPTO binding partner. There is no evidence for the O-linked fucose modification in any of the soluble EGF-ligands, i.e. epidermal growth factor, transforming growth factor alpha, heregulin.
From a diagnostic or therapeutic perspective, there is considerable interest for the development of CRIPTO mutants with desirable anti-carcinogenic properties.